1. Field of the Invention
This invention relates generally to methods and apparatus employing a plurality of transfer elements for multiplexing transfer of droplets of sample from multiwell source plates to surfaces reservoirs such as in miniaturized cassettes, in which chemical syntheses and analyses are possible. The invention is useful for the generation of combinatorial libraries and high throughput screening in, for example, pharmaceutical drug discovery, agricultural pesticide discovery, genomic science applications and the like.
2. Description of the Related Art
In a range of technology-based business sectors, including the chemical, bioscience, biomedical, and pharmaceutical industries, it has become increasingly desirable to develop capabilities for rapidly and reliably carrying out chemical and biochemical reactions in large numbers using small quantities of samples and reagents. Carrying out a massive screening program manually, for example, can be exceedingly time consuming and may be entirely impracticable where only a very small quantity of an important sample or component of interest is available, or where a component of a synthesis or analysis is very costly.
Accordingly, considerable resources have been directed to developing methods for high-throughput chemical syntheses, screening, and analyses. Considerable art has emerged, in part, from such efforts.
Automated laboratory workstations have contributed significantly to advances in pharmaceutical drug discovery and genomic science over the past decade. See for example, U.S. Pat. Nos. 5,104,621 and 5,356,525 (Beckman Instruments). More specifically, robotics technology has played a major role in providing a practical useful means for enabling high throughput screening (HTS) methods. Reference can be made, for example, to U.S. Pat. No. 4,965,049.
In addition to the emergence of automation technology, the last decade has seen an enormous advance in the scientific understanding of critical cellular processes, and this has led to rationally designed approaches in drug discovery. Also, the application of molecular genetics and recombinant DNA technology, U.S. Pat. No. 4,237,224 (Cohen and Boyer), has led to the isolation of many genes encoding proteins, which show promise as targets for new drugs. Once a target gene is identified, the recombinant protein can be heterologously expressed in mammalian tissue culture cells, insect cells, bacteria and/or, yeast.
The advantages of employing molecular cloning techniques are many. Often receptors and enzymes exist in alternative forms, subtypes or isoforms. Using a cloned target focuses the primary screen on the subtype appropriate for the disease. Agonists or antagonists can be identified and their selectivity can then be tested against the other known subtypes. The availability of such cloned genes and corresponding expression systems have enabled new types of screens to be created that are specific, sensitive, and often automatable.
Matched with the scientific and technological advances in biology has been the emergence of innovative methods for highly parallel chemical synthesis. For several decades, preparation of synthetic analogs to the prototypic lead compound was the established method for drug discovery. Natural products were usually isolated from soil microbes and cultured under a wide variety of conditions. The spectrum of organisms employed by the pharmaceutical industry for isolation of natural products has now expanded from Actinomycetes and fungi to include plants, marine organisms, and insects.
During the last five years, the chemistry of creating combinatorial libraries has made a vastly increased number of synthetic compounds available for testing. More specifically, thousands to tens or hundreds of thousands of small molecules can be rapidly and economically synthesized. See, for example, U.S. Pat. No. 5,252,743 (Affymax Technologies N.V.) for a discussion of combinatorial chemistry. Thus, combinatorial libraries complement the large numbers of synthetic compounds available from the more traditional drug discovery programs based, in part, on identifying lead compounds through natural product screening.
Competitive binding assays, originally developed in the 1960's for immunodiagnostic applications, continue to be commonly employed for quantitatively characterizing receptor-ligand interactions. Despite advances in the development of spectrophotometric and fluorometric-based bioanalytical assays, radiolabeled ligands are still commonly employed in pharmaceutical HTS applications. Although non-isotopic markers promise to be environmentally cleaner, safer, less expensive, and generally easier to use than radioactive compounds, sensitivity limitations have prevented these new methods from becoming widespread. Another major disadvantage of the competition assay is the number of steps, most notably, washing steps, required to run the assays.
A few years ago, Scintillation Proximity Assays were introduced by Amersham and also are discussed in U.S. Pat. Nos. 4,271,139 and 4,382,074 as a means of circumventing the wash steps required in the above heterogeneous assays. The new homogeneous assay technology, which requires no separation of bound from free ligand, is based on the coating of scintillant beads with an acceptor molecule, for example, the target receptor.
Another variation of this theme avoids the use of radioactivity and is especially useful in high-throughput assays. The modification involves the use of lanthanide chelates in time-resolved fluorometry. Aspects of this particular homogeneous assay technology are discussed in U.S. Pat. No. 5,637,509. This particular technology takes advantage of the unique properties of the lanthanide chelate europium-cryptate in combination with the energy absorbing molecule, allophycocyanin (APC).
Robotic-based high-throughput tools are now routinely used for screening libraries of compounds for the purpose of identifying lead molecules for their therapeutic potential. Subsequently, considerable art has emerged. For example, a screening method for characterizing ligand binding to a given target employing a variety of separation techniques is described in the PCT application WO 97/01755. Another related method is described in U.S. Pat. No. 5,585,277 (Scriptgen Pharmaceuticals).
Highly parallel and automated methods for DNA synthesis and sequencing have also contributed significantly to the success of the human genome project to date. For example, PE/Applied Biosystems (ABI), PerSeptive BioSystems, Pharmacia Biotech, and Beckman Instruments have developments in DNA synthesis instrumentation. In the area of DNA sequencing, ABI and LiCor are active. In addition, see U.S. Pat. No. 5,455,008. For a related invention, see Genzyme Corporation's HTS method for DNA analysis that is described in U.S. Pat. No. 5,589,330. For sequencing by hybridization, see PCT WO 89/10977 (Southern), Affymetrix (U.S. Pat. Nos. 5,599,695 and 5,631,734), and U.S. Pat. No. 5,202,231 (Drmanac, et al.).
Computerized data handling and analysis systems have also emerged with the commercial availability of high-throughput instrumentation for numerous life sciences research and development applications. Commercial software, including database and data management software, has become routine in order to efficiently handle the large amount of data being generated. Bioinformatics has emerged as an important field.
With the developments outlined above in molecular and cellular biology, combined with advancements in combinatorial chemistry, there has been an exponential increase in the number of targets and compounds available for screening. In addition, many new genes and their expressed proteins will be identified by the Human Genome project and will therefore greatly expand the pool of new targets for drug discovery. Subsequently, an unprecedented interest has arisen in the development of more efficient ultrahigh throughput methods and instrumentation for pharmaceutical and genomic science screening applications.
In recent parallel technological developments, miniaturization of chemical analysis systems, employing semiconductor processing methods, including photolithography and other wafer fabrication techniques borrowed from the microelectronics industry, has attracted increasing attention and has progressed rapidly. The so-called "lab-on-a-chip" technology enables sample preparation and analysis to be carried out on-board microfluidic-based cassettes. Moving fluids through a network of interconnecting enclosed microchannels of capillary dimensions is possible using electrokinetic transport methods.
Application of microfluidics technology embodied in the form of analytical devices has many attractive features for pharmaceutical high throughput screening. Advantages of miniaturization include greatly increased throughput and reduced costs, in addition to low consumption of both sample and reagents and system portability. Implementation of these developments in microfluidics and laboratory automation hold great promise for contributing to advancements in life sciences research and development.
Nonetheless, the 96 well microtiter plate and multiples thereof such as, e.g., the 384 well microtiter plate, have been, and still are, the pharmaceutical industry standard for carrying out bioanalytical assays despite the recent advances in miniaturization and microfluidics. Because an enormous number of synthetic libraries have been, and continue to be, generated using this particular multiwell format, the microtiter plate will remain entrenched within the industry.
As microfluidic technologies advance, new methods for enabling fluid transfer between multi-well plates and microassay cassettes would be beneficial. A critical factor currently limiting such a microfluidic HTS hybrid device is a means for reproducible liquid communication between the disparate dimensions of the two systems. More specifically, integration of microfluidics technology with existing robotic-based methods currently used in automated workstations is constrained by differences in volume size of samples used. For these reasons, new automated methods for multiplexing common lab tasks such as sample handling and dispensing on the microscale are required. Once again, other parallel developments, in this case borrowed from the ink jet printing industry, are applicable to fulfilling, at least in part, this currently unmet technological need. The art is briefly reviewed.
Various droplet ejector technologies have been or are being developed. One such technology, electrostatic discharge, is commonly used for dispensing fluids and reference may be made to U.S. Pat. Nos. 4,749,125; 5,086,973; 5,165,601 issued to Terronics Development Corp.; and U.S. Pat. No. 5,332,154 to Lundy and Associates.
Other devices use electrostatic energy to eject ink onto a recording medium. For a more detailed description of electrostatic ink printing, reference may be made to U.S. Pat. No. 5,588,597 to MicroParts GmbH; U.S. Pat. No. 5,278,583 to Matsushita Electric Industrial Co.; U.S. Pat. No. 4,915,718 to On Target Technology, Inc.; and U.S. Pat. No. 4,799,068 to Fuji Xerox Co., Ltd.
Another related invention includes Quate's acoustic fluid ejector system as described in U.S. Pat. No. 5,608,433 issued to Xerox Corp. Other related U.S. Patents include U.S. Pat. No. 5,586,723 issued to Spraying Systems Co.; U.S. Pat. No. 5,164,740 issued to Yehuda Ivr, and the citations therein.
Another ejector technology, piezoelectric ejection, is discussed in U.S. Pat. No. 5,164,740. For a more detailed description of piezoelectric printing, reference may be made to U.S. Pat. No. 5,529,055 issued to L'Oreal and the citations therein.
An apparatus for liquid transfer has been made and used for delivering a plurality of samples in sequence to treatment reservoirs wherein a chemical reaction or physical treatment step occurs. See, e.g., U.S. Pat. No. 5,188,148 for a conduit plate for fluid delivery system and U.S. Pat. No. 5,325,889 for a laminated conduit plate for fluid delivery system (both issued to Millipore Corp.).
Aspiration devices involve pneumatic forces or back pressure for its mechanism of action. See, e.g., U.S. Pat. No. 5,463,910 for a multi-function aspirating device (AVL Scientific Corp.); U.S. Pat. No. 5,384,093 for an apparatus for aspirating and discharging a liquid sample (Toa Medical Electronics Co., Ltd.); and U.S. Pat. No. 5,525,302 for a method and device for simultaneously transferring plural samples.
A multiwell plate is disclosed in PCT WO 97/15394 published May 1, 1997 (SmithKline Beecham Corporation). The wells have a large opening at the top and small nozzle hole in the base. The opening is chosen so that a jet of liquid is emitted when a pressure pulse is applied to the surface such that by selecting a time for the pressure pulse a precise amount of volume in the well can be dispensed.